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Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcohol‐associated and metabolic‐associated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcohol‐associated liver disease plus metabolic‐associated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNA‐sequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets.

Binge drinking, i.e., heavy episodic drinking in a short time, has recently become an alarming societal problem with negative health impact. However, the harmful effects of acute alcohol injury in the gut-liver axis remain elusive. Hence, we focused on the physiological and pathological changes and the underlying mechanisms of experimental binge drinking in the context of the gut-liver axis. Eight-week-old mice with a C57BL/6 background received a single dose (p.o.) of ethanol (EtOH) [6 g/kg b.w.] as a preclinical model of acute alcohol injury. Controls received a single dose of PBS. Mice were sacrificed 8 h later. In parallel, HepaRGs and Caco-2 cells, human cell lines of differentiated hepatocytes and intestinal epithelial cells intestinal epithelial cells (IECs), respectively, were challenged in the presence or absence of EtOH [0–100 mM]. Extracellular vesicles (EVs) isolated by ultracentrifugation from culture media of IECs were added to hepatocyte cell cultures. Increased intestinal permeability, loss of zonula occludens-1 (ZO-1) and MUCIN-2 expression, and alterations in microbiota—increased Lactobacillus and decreased Lachnospiraceae species—were found in the large intestine of mice exposed to EtOH. Increased TUNEL-positive cells, infiltration of CD11b-positive immune cells, pro-inflammatory cytokines (e.g., tlr4, tnf, il1β), and markers of lipid accumulation (Oil Red O, srbep1) were evident in livers of mice exposed to EtOH, particularly in females. In vitro experiments indicated that EVs released by IECs in response to ethanol exerted a deleterious effect on hepatocyte viability and lipid accumulation. Overall, our data identified a novel mechanism responsible for driving hepatic injury in the gut-liver axis, opening novel avenues for therapy.

Idiosyncratic drug-induced liver injury (DILI) is a type of hepatic injury caused by an uncommon drug adverse reaction that can develop to conditions spanning from asymptomatic liver laboratory abnormalities to acute liver failure (ALF) and death. The cellular and molecular mechanisms involved in DILI are poorly understood. Hepatocyte damage can be caused by the metabolic activation of chemically active intermediate metabolites that covalently bind to macromolecules (e.g., proteins, DNA), forming protein adducts—neoantigens—that lead to the generation of oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress, which can eventually lead to cell death. In parallel, damage-associated molecular patterns (DAMPs) stimulate the immune response, whereby inflammasomes play a pivotal role, and neoantigen presentation on specific human leukocyte antigen (HLA) molecules trigger the adaptive immune response. A wide array of antioxidant mechanisms exists to counterbalance the effect of oxidants, including glutathione (GSH), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), which are pivotal in detoxification. These get compromised during DILI, triggering an imbalance between oxidants and antioxidants defense systems, generating oxidative stress. As a result of exacerbated oxidative stress, several danger signals, including mitochondrial damage, cell death, and inflammatory markers, and microRNAs (miRNAs) related to extracellular vesicles (EVs) have already been reported as mechanistic biomarkers. Here, the status quo and the future directions in DILI are thoroughly discussed, with a special focus on the role of oxidative stress and the development of new biomarkers.

Background: The development of obesity-associated comorbidities such as type 2 diabetes (T2D) and hepatic steatosis has been linked to selected microRNAs in individual studies; however, an unbiased genome-wide approach to map T2D induced changes in the miRNAs landscape in human liver samples, and a subsequent robust identification and validation of target genes are still missing. Methods: Liver biopsies from age- and gender-matched obese individuals with (n=20) or without (n=20) T2D were used for microRNA microarray analysis. The candidate microRNA and target genes were validated in 85 human liver samples, and subsequently mechanistically characterized in hepatic cells as well as by dietary interventions and hepatic overexpression in mice. Results: Here, we present the human hepatic microRNA transcriptome of type 2 diabetes in liver biopsies and use a novel seed prediction tool to robustly identify microRNA target genes, which were then validated in a unique cohort of 85 human livers. Subsequent mouse studies identified a distinct signature of T2D-associated miRNAs, partly conserved in both species. Of those, human-murine miR-182–5 p was the most associated with whole-body glucose homeostasis and hepatic lipid metabolism. Its target gene LRP6 was consistently lower expressed in livers of obese T2D humans and mice as well as under conditions of miR-182–5 p overexpression. Weight loss in obese mice decreased hepatic miR-182–5 p and restored Lrp6 expression and other miR-182–5 p target genes. Hepatic overexpression of miR-182–5 p in mice rapidly decreased LRP6 protein levels and increased liver triglycerides and fasting insulin under obesogenic conditions after only seven days. Conclusions: By mapping the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, validating conserved miRNAs in diet-induced mice, and establishing a novel miRNA prediction tool, we provide a robust and unique resource that will pave the way for future studies in the field. As proof of concept, we revealed that the repression of LRP6 by miR-182–5 p, which promotes lipogenesis and impairs glucose homeostasis, provides a novel mechanistic link between T2D and non-alcoholic fatty liver disease, and demonstrate in vivo that miR-182–5 p can serve as a future drug target for the treatment of obesity-driven hepatic steatosis. Funding: This work was supported by research funding from the Deutsche Forschungsgemeinschaft (KI 1887/2-1, KI 1887/2-2, KI 1887/3-1 and CRC-TR296), the European Research Council (ERC, CoG Yoyo LepReSens no. 101002247; PTP), the Helmholtz Association (Initiative and Networking Fund International Helmholtz Research School for Diabetes; MB) and the German Center for Diabetes Research (DZD Next Grant 82DZD09D1G).

Abstract Mutations in thyroid hormone receptor α1 (TRα1) cause Resistance to Thyroid Hormone α (RTHα), a disorder characterized by hypothyroidism in TRα1-expressing tissues including the heart. Surprisingly, we report that treatment of RTHα patients with thyroxine to overcome tissue hormone resistance does not elevate their heart rate. Cardiac telemetry in male, TRα1 mutant, mice indicates that such persistent bradycardia is caused by an intrinsic cardiac defect and not due to altered autonomic control. Transcriptomic analyses show preserved, thyroid hormone (T3)-dependent upregulation of pacemaker channels (Hcn2, Hcn4), but irreversibly reduced expression of several ion channel genes controlling heart rate. Exposure of TRα1 mutant male mice to higher maternal T3 concentrations in utero, restores altered expression and DNA methylation of ion channels, including Ryr2. Our findings indicate that target genes other than Hcn2 and Hcn4 mediate T3-induced tachycardia and suggest that treatment of RTHα patients with thyroxine in high dosage without concomitant tachycardia, is possible.

Summary: Agonists for thyroid hormone receptor β (TRβ) show promise in preclinical studies and clinical trials to improve non-alcoholic fatty liver disease. A recent study on human livers, however, revealed reduced TRβ expression in non-alcoholic steatohepatitis (NASH), indicating a developing thyroid hormone resistance, which could constitute a major obstacle for those agonists.Using a rapid NASH paradigm combining choline-deficient high-fat diet and thermoneutrality, we confirm that TRβ declines during disease progression in mice similar to humans. Contrary to expectations, mice lacking TRβ showed less liver fibrosis, and NASH marker genes were not elevated. Conversely, increasing TRβ expression in wild-type NASH mice using liver-targeted gene therapy did not improve histology, gene expression, or metabolic parameters, indicating that TRβ receptor levels are of minor relevance for NASH development and progression in our model, and suggest that liver—rather than isoform—specificity might be more relevant for NASH treatment with thyroid hormone receptor agonists.